Method for determining nucleotide identity through primer extension

- Molecular Tool, Inc.

The invention concerns a reagent composition that employs at least two different terminators of a nucleic acid template-dependent primer extension reaction to determine the identity of a nucleotide base at a specific position in a nucleic acid of interest. The invention also concerns the method for determining such identification. The invention may be used to determine the presence or absence of a specific nucleotide sequence in a sample. It may also be employed in determination of genotype and in the identification of different alleles.

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Claims

1. A method of determining the identity of a nucleotide base at a specific position in a nucleic acid of interest, which comprises:

(a) treating a sample containing the nucleic acid of interest, if the nucleic acid is double-stranded, so as to obtain unpaired nucleotide bases spanning the specific position, or directly employing step (b) if the nucleic acid of interest is single-stranded;
(b) contacting the sample from step (a), with an oligonucleotide primer which is fully complementary to and which hybridizes specifically to a stretch of nucleotide bases present in the nucleic acid of interest immediately adjacent to the nucleotide base to be identified, under high stringency hybridization conditions, so as to form a duplex between the primer and the nucleic acid of interest such that the nucleotide base to be identified is the first unpaired base in the template immediately downstream of the 3' end of the primer in said duplex; and
(c) contacting the duplex from step (b), in the absence of dATP, dCTP, dGTP, or dTTP, with at least two different terminators of a nucleic acid template-dependent, primer extension reaction capable of specifically terminating the extension reaction in a manner strictly dependent upon the identity of the unpaired nucleotide base in the template immediately downstream of the 3' end of the primer wherein one of said terminators is complementary to said nucleotide base to be identified and wherein at least one of said terminators is labeled with a detectable marker; wherein said contacting is under conditions sufficient to permit base pairing of said complementary terminator with the nucleotide base to be identified and occurrence of a template-dependent primer extension reaction sufficient to incorporate said complementary terminator onto the 3' end of the primer to thereby extend said 3' end of said primer by one terminator;
(d) determining the presence and identity of the nucleotide base at the specific position in the nucleic acid of interest by detecting the detectable marker of said incorporated terminator while said terminator is incorporated at the 3' end of the extended primer, and wherein said detection is conducted in the absence of non-terminator nucleotides.

2. The method of claim 1, wherein in step (c), the duplex from step (b) is contacted with four labeled terminators, wherein only one of the terminators has a detectable marker, and wherein in said method said step (c) is performed four times, with a different one of the four terminators being labelled.

3. The method of claim 1, wherein in step (c), the duplex from step (b) is contacted with four labeled terminators, each with a different detectable label.

4. A method of typing a sample containing nucleic acids which comprises identifying the nucleotide base or bases present at each of one or more specific positions, each such nucleotide base being identified using the method of claim 2 or 3, and each such specific position being determined using a different primer.

5. A method of claim 4, wherein the identity of each nucleotide base or bases at each position is determined individually or wherein the identities of the nucleotide bases at different positions are determined simultaneously.

6. A method for identifying different alleles in a sample containing nucleic acids which comprises identifying the nucleotide base or bases present at each of one or more specific positions, each such nucleotide base being identified by the method of claim 2 or 3.

7. A method for determining the genotype of an organism at one or more particular genetic loci which comprises:

(a) obtaining from the organism a sample containing genomic DNA; and
(b) identifying the nucleotide base or bases present at each of one or more specific positions in nucleic acids of interest, each such base or bases being identified using the method of claim 2 or 3, and thereby identifying different alleles and thereby, in turn, determining the genotype of the organism at one or more particular genetic loci.

8. A method of claim 2 or 3, wherein the conditions for the occurrence of the template-dependent, primer extension reaction in step (c) are created, in part, by the presence of a suitable template-dependent enzyme.

9. A method of claim 8, wherein the template-dependent enzyme is E. coli DNA polymerase I or the "Klenow fragment" thereof, T4 DNA polymerase, T7 DNA polymerase T. aquaticus DNA polymerase, a retroviral reverse transcriptase, or combinations thereof.

10. A method of claim 2 or 3, wherein the nucleic acid of interest is a deoxyribonucleic acid, a ribonucleic acid, or a copolymer of deoxyribonucleic acid and ribonucleic acid.

11. A method of claim 2 or 3, wherein the primer is an oligodeoxyribonucleotide, an oligoribonucleotide, or a copolymer of deoxyribonucleic acid and ribonucleic acid.

12. A method of claim 2 or 3, wherein the template is a deoxyribonucleic acid, the primer is an oligodeoxyribonucleotide, oligoribonucleotide, or a copolymer of deoxyribonucleotides and ribonucleotides, and the template-dependent enzyme is a DNA polymerase.

13. A method of claim 2 or 3, wherein the template is a ribonucleic acid, the primer is an oligodeoxyribonucleotide, oligoribonucleotide, or a copolymer of deoxyribonucleotides and ribonucleotides, and the template-dependent enzyme is a reverse transcriptase.

14. A method of claim 2 or 3, wherein the template is a deoxyribonucleic acid, the primer is an oligoribonucleotide, and the enzyme is an RNA polymerase.

15. A method of claim 2 or 3, wherein the template is a ribonucleic acid, the primer is an oligoribonucleotide, and the template-dependent enzyme is an RNA replicase.

16. A method of claim 2 or 3, wherein, prior to the primer extension reaction in step (c), the template has been capped at its 3' end by the addition of a terminator to the 3' end of the template, said terminator being capable of terminating a template-dependent, primer extension reaction.

17. A method of claim 16, wherein the terminator is a dideoxynucleotide.

18. A method of claim 2 or 3, wherein the nucleic acid of interest has been synthesized enzymatically in vivo, synthesized enzymatically in vitro, or synthesized non-enzymatically.

19. A method of claim 2 or 3, wherein the oligonucleotide primer has been synthesized enzymatically in vivo, synthesized enzymatically in vitro, or synthesized non-enzymatically.

20. A method of claim 2 or 3, wherein the oligonucleotide primer comprises one or more moieties that permit affinity separation of the primer from the unincorporated reagent and/or the nucleic acid of interest.

21. A method of claim 20, wherein the oligonucleotide primer comprises biotin which permits affinity separation of the primer from the unincorporated reagent and/or nucleic acid of interest via binding of the biotin to streptavidin which is attached to a solid support.

22. A method of claim 2 or 3, wherein the sequence of the oligonucleotide primer comprises a DNA sequence that permits affinity separation of the primer from the unincorporated reagent and/or the nucleic acid of interest via base pairing to a complementary sequence present in a nucleic acid attached to a solid support.

23. A method of claim 2 or 3, wherein the nucleic acid of interest comprises one or more moieties that permit affinity separation of the nucleic acid of interest from the unincorporated reagent and/or the primer.

24. A method of claim 23, wherein the nucleic acid of interest comprises biotin which permits affinity separation of the nucleic acid of interest from the unincorporated reagent and/or the primer via binding of the biotin to streptavidin which is attached to a solid support.

25. A method of claim 2 or 3, wherein the sequence of the nucleic acid of interest comprises a DNA sequence that permits affinity separation of the nucleic acid of interest from the unincorporated reagent and/or the primer via base pairing to a complementary sequence present in a nucleic acid attached to a solid support.

26. A method of claim 2 or 3, wherein the oligonucleotide primer is labeled with a detectable marker.

27. A method of claim 26, wherein the oligonucleotide primer is labeled with a detectable marker that is different from any detectable marker present in the reagent or attached to the nucleic acid of interest.

28. A method of claim 2 or 3, wherein the nucleic acid of interest is labeled with a detectable marker.

29. A method of claim 28, wherein the nucleic acid of interest is labeled with a detectable marker that is different from any detectable marker present in the reagent or attached to the primer.

30. A method of claim 2 or 3, wherein the nucleic acid of interest comprises non-natural nucleotide analogs.

31. A method of claim 30, wherein the non-natural nucleotide analogs comprise deoxyinosine or 7-deaza-2'-deoxyguanosine.

32. A method of claim 2 or 3, wherein the nucleic acid of interest has been synthesized by the polymerase chain reaction.

33. A method of claim 2 or 3, wherein the sample comprises genomic DNA from an organism, RNA transcripts thereof, or cDNA prepared from RNA transcripts thereof.

34. A method of claim 2 or 3, wherein the sample comprises extragenomic DNA from an organism, RNA transcripts thereof, or cDNA prepared from RNA transcripts thereof.

35. A method of claim 2 or 3, wherein the primer is separated from the nucleic acid of interest after the primer extension reaction in step (c) by using appropriate denaturing conditions.

36. A method of claim 35, wherein the denaturing conditions comprise heat, alkali, formamide, urea, glyoxal, enzymes, and combinations thereof.

37. A method of claim 36, wherein the denaturing conditions comprise treatment with 0.2N NaOH.

38. A method of claim 33, wherein the organism is a plant, microorganism, virus, or bird.

39. A method of claim 33, wherein the organism is a vertebrate or invertebrate.

40. A method of claim 33, wherein the organism is a mammal.

41. A method of claim 40, wherein the mammal is a human being.

42. A method of claim 40, wherein the mammal is a horse, dog, cow, cat, pig, or sheep.

43. A method of determining the presence or absence of a particular target nucleic acid molecule in a sample of nucleic acids, which comprises:

(a) treating the sample of nucleic acids, if such sample of nucleic acids contains double-stranded nucleic acids, so as to obtain single-stranded nucleic acids, or directly employing step (b) if the sample of nucleic acids contains only single-stranded nucleic acids;
(b) contacting the sample from step (a), with an oligonucleotide primer which is fully complementary to and which hybridizes specifically to a stretch of nucleotide bases present in said target nucleic acid molecule immediately adjacent to a preselected nucleotide base, under high stringency hybridization conditions so as to form a duplex between the primer and the nucleic acid of interest such that the preselected nucleotide base is the first unpaired base in the template immediately downstream of the 3' end of the primer in said duplex; and
(c) contacting the duplex from step (b), in the absence of dATP, dCTP, dGTP, or dTTP, with at least two different terminators of a nucleic acid template-dependent, primer extension reaction capable of specifically terminating the extension reaction in a manner strictly dependent upon the identity of the unpaired nucleotide base in the template immediately downstream of the 3' end of the primer wherein one of said terminators is complementary to said nucleotide base to be identified and wherein at least one of said terminators is labeled with a detectable marker; wherein said contacting is under conditions sufficient to permit base pairing of said complementary terminator with said unpaired nucleotide base immediately downstream of the 3' end of said hybridized primer and occurrence of a template-dependent primer extension reaction sufficient to incorporate said complementary terminator onto the 3' end of the primer to thereby extend said 3' end of said primer by one terminator;
(d) determining the presence of the target nucleic acid molecule by detecting the detectable marker of said incorporated terminator while said terminator is incorporated at the 3' end of the extended primer, and wherein said detection is conducted in the absence of non-terminator nucleotides.

44. The method of claim 43, wherein in step (c), the duplex from step (b) is contacted with four labeled terminators, wherein only one of the terminators has a detectable marker, and wherein in said method said step (p) is performed four times, with a different one of the four terminators being labelled.

45. The method of claim 43, wherein in step (c), the duplex from step (b) is contacted with four labeled terminators, each with a different detectable label.

46. A method of determining the identity of a nucleotide base at a specific position in a nucleic acid of interest which comprises:

(a) (1) incubating a sample containing the nucleic acid of interest with at least two oligonucleotide primers, and a polymerase, said primers being sufficient to mediate a polymerase chain reaction amplification of said nucleic acid of interest, wherein said incubation is conducted under conditions sufficient to permit said amplification to occur;
(2) treating a sample containing said amplified nucleic acid of interest, if such nucleic acid is double-stranded, so as to obtain unpaired nucleotide bases spanning the specific position, or directly employing step (b) if the nucleic acid of interest is single-stranded;
(b) contacting the sample from step (a2), under hybridizing conditions, with an oligonucleotide primer which is fully complementary to and which hybridizes specifically to a stretch of nucleotide bases present in the nucleic acid of interest immediately adjacent to the nucleotide base to be identified, so as to form a duplex between the primer and the nucleic acid of interest such that the nucleotide base to be identified is the first unpaired base in the template immediately downstream of the 3' end of the primer in said duplex; and
(c) contacting the duplex from step (b), in the absence of dATP, dCTP, dGTP, or dTTP, with at least two different terminators of a nucleic acid template-dependent, primer extension reaction capable of specifically terminating the extension reaction in a manner strictly dependent upon the identity of the unpaired nucleotide base in the template nucleic acid of interest immediately downstream of the 3' end of the primer wherein one of said terminators is complementary to said nucleotide base to be identified and wherein at least one of said terminators is labeled with a detectable marker, wherein said contacting is under conditions sufficient to permit base pairing of said complementary terminator with the nucleotide base to be identified and occurrence of a template-dependent primer extension reaction sufficient to incorporate said complementary terminator onto the 3' end of the primer to thereby extend said 3' end of said primer by one terminator;
(d) determining the presence and identity of the nucleotide base at the specific position in the nucleic acid of interest by detecting the detectable market of said incorporated terminator while said terminator is incorporated at the 3' end of the extended primer, and wherein said detection is conducted in the absence of non-terminator nucleotides.

47. A method of determining the presence or absence of a particular target nucleic acid molecule in a sample of nucleic acids which comprises:

(a) (1) incubating said sample of nucleic acids with at least two oligonucleotide primers, and a polymerase, said primers being sufficient to mediate a polymerase chain reaction amplification of said desired polynucleotide if said desired polynucleotide is present in said sample, wherein said incubation is conducted under conditions sufficient to permit said amplification to occur;
(2) treating said incubated sample of nucleic acids, if such sample of nucleic acids contains double-stranded nucleic acids, so as to obtain single-stranded nucleic acids, or directly employing step (b) if the sample of nucleic acids contains only double-stranded nucleic acids;
(b) contacting the sample from step (a2), under hybridizing conditions, with an oligonucleotide primer which is fully complementary to and which hybridizes specifically to a stretch of nucleotide bases present in said target nucleic acid molecule immediately adjacent to a preselected nucleotide base, so as to form a duplex between the primer and the nucleic acid of interest such that the preselected nucleotide base is the first unpaired base in the template immediately downstream of the 3' end of the primer in said duplex; and
(c) contacting the duplex from step (b), in the absence of dATP, dCTP, dGTP, or dTTP, with at least two different terminators of a nucleic acid template-dependent, primer extension reaction capable of specifically terminating the extension reaction in a manner strictly dependent upon the identity of the unpaired nucleotide base in the template immediately downstream of the 3' end of the primer wherein one of said terminators is complementary to said nucleotide base to be identified and wherein at least one of said terminators is labeled with a detectable marker; wherein said contacting is under conditions sufficient to permit base pairing of said complementary terminator with said unpaired nucleotide base immediately downstream of the 3' end of said hybridized primer and occurrence of a template-dependent, primer extension reaction sufficient to incorporate said complementary terminator onto the 3' end of the primer to thereby extend said 3' end of said primer by one terminator; and
(d) determining the presence of the target nucleic acid molecule by detecting the detectable marker of said incorporated terminator while said terminator is incorporated at the 3' end of the extended primer, and wherein said detection is conducted in the absence of non-terminator nucleotides.

48. A method of typing a sample containing nucleic acids which comprises determining the presence or absence of one or more particular nucleotide sequences, the presence or absence of each such nucleotide sequence being determined by the method of claim 40 or 47.

Referenced Cited

U.S. Patent Documents

4656127 April 7, 1987 Mundy
4962020 October 9, 1990 Tabor et al.

Other references

  • K. B. Mullis, Scientific American, (Apr. 1990), "The Unusual Origin of the Polymerase Chain Reaction", pp. 56-65. Prober et al. (1987) Science, vol. 238, pp. 336-341.

Patent History

Patent number: 5888819
Type: Grant
Filed: Mar 5, 1991
Date of Patent: Mar 30, 1999
Assignee: Molecular Tool, Inc. (Baltimore, MD)
Inventors: Philip Goelet (Cockeysville, MD), Michael R. Knapp (Baltimore, MD), Stephen Anderson (Princeton, NJ)
Primary Examiner: Lisa B. Arthur
Attorney: Howrey & Simon
Application Number: 7/664,837

Classifications

Current U.S. Class: Involving Virus Or Bacteriophage (435/5); 435/6; Absidia (435/912)
International Classification: C12Q 170; C12Q 168; C12P 1934;